Crystal structures of herbicide-detoxifying esterase reveal a lid loop affecting substrate binding and activity

SulE, an esterase, which detoxifies a variety of sulfonylurea herbicides through de-esterification, provides an attractive approach to remove environmental sulfonylurea herbicides and develop herbicide-tolerant crops. Here, we determined the crystal structures of SulE and an activity improved mutant P44R. Structural analysis revealed that SulE is a dimer with spacious binding pocket accommodating the large sulfonylureas substrate. Particularly, SulE contains a protruding β hairpin with a lid loop covering the active site of the other subunit of the dimer. The lid loop participates in substrate recognition and binding. P44R mutation altered the lid loop flexibility, resulting in the sulfonylurea heterocyclic ring repositioning to a relative stable conformation thus leading to dramatically increased activity. Our work provides important insights into the molecular mechanism of SulE, and establish a solid foundation for further improving the enzyme activity to various sulfonylurea herbicides through rational design.

The paper is well written, although some minor English errors occur throughout and these would need to be corrected. For instance on Line 107 singular form of the verb to be (is) should be plural (are). Line 303 -has not was, Line 344 should read -Previous results.
There are also some inconsistencies regarding the name of the related esterase enzyme. On Line 105 the protein 1QLW is called esterase 731, while on Line 223, 226, 231 it is called esterase 713. Table 1 would benefit from inclusion of Wilson B-factor of each data set and the clashscore of each structure.
The authors claim that there are features of their reported esterase -absence of the signature sequence around the active site serine and location of the acidic residue (GLU) from the active site triad on a different secondary structure element. These however are not unique and they are also present in pdb structures 1QLW and 4Q34. These features have been reported in detail previously in a paper in 2000 in the case of 1QLM and a structure has been deposited in the PDB 4Q34 from a structural genomics project -see below. This structure should also be discussed in this submitted manuscript. The structure reported in the submitted paper appears to be a different member of this esterase family and the reference to 1QLW should be mentioned in the introduction when the similarity is first mentioned.
Crystal structure of a putative esterase (BDI_1566) from Parabacteroides distasonis ATCC 8503 at 1.60 A resolution Joint Center for Structural Genomics (JCSG) PDB 4Q34 The Atomic Resolution Structure of a Novel Bacterial Esterase Bourne, P.C., Isupov, M.N., Littlechild, J.A. (2000) Structure 8: 143 PDB 1QLW Its ability of the reported enzyme to be used for sulfonylurea herbicide detoxification is interesting together with the mutant enzyme with improved activity.
Reviewer #2 (Remarks to the Author): In the manuscript, Liu et al characterized an esterase which detoxicates and hydrolyses a variety of sulfonylurea herbicides. They reported several crystal structures of SulE, in its apo form (WT and P44R) or complexed with CA (WT, P44R/S209A) and MM (S209A). Their crystal structures revealed a lid loop in a domain-swapped β-hairpin covering the active site from the opposing monomer. Based on their previous finding of an improved mutant P44R (previously annotated as P80R) from the lid loop region, they found the flexibility of the loop modulates substrate binding and enzyme activity and provided structural and mutational evidence. Determination of complex structures of SulE with substrate MM and product CA (hydrolysed from CE) did provide useful insight into the substrate binding and catalytic mechanism during catalysis. The observation of lid loop shift in complex structure of P44R-S209A-CA compared to the S209A-CA complex is one major novel finding which indicates the flexibility of the lid loop contributes to catalysis. However, the manuscript is not well written and presented. It needs further proof reading to improve grammar and reference citation. The interpretation of the crystal structures is bit of shallow and problematic in some part.
The authors observed residual activity for S209A. It is indeed very uncommon and hard to imagine mechanically. Ser209 is the nucleophile to attack the ester carbonyl to form the acyl-enzyme tetrahedral intermediate. S209A is unable to perform the attack and thus would be impossible to form the acyl-enzyme intermediate. The authors explained this result with a water as the possible nucleophile for S209A. Have the authors seen any water in hydrogen bonding distance with the ester carbonyl in S209A mutant structure?
It's not clear in this manuscript whether residual activity has been seen for S209A with MM or other substrates? They saw MM in the S209A complex structure but, they saw hydrolysed CA complex structure for CE substrate (although a CE is found in the dimer interface). MM is a better substrate for wild type SulE (2-fold) than CE (J. Agric. Food Chem. 2019, 67, 3, 836-843). My other suggestion is that, to obtain a clean fully occupied structure of CA complex, a hydrolysed product for co-crystallization is a better choice or to obtain a substrate complex, do soaking in the presence of high concentration of substrate or use a R150A or S209A/H333A for co-crystallization. Show the density in separate panels. Ala210 is hidden behind the density. In Panel D, it's hard to tell which mutant is dead or severely impaired (G78A, R150A, S209A, E232A, H333A, S209A/H333A) from the figure. Adjust the y-scale for some mutants to show the difference. Figure 5A. Colour Tyr45 differently in apo P44R/S209A or CA bound complex structures. It's hard to tell which conformation belongs to apo or CA bound.
Line 158-160. "As expected, mutation of Ser209, Glu232 or His333 to Ala almost completely abolished the de-esterification activity (about 0.001% activity retained), and the double mutation of Ser209 and His333 resulted in a complete loss of esterase activity ( Figure 3D), …". They authors should emphasize which substrate was used in this assay or do they all show residual activity? [203][204][205]The ester O atom of MM is hydrogen-bonded with the main chain nitrogen atoms of Gly78 and Ala210, forming an oxyanion hole." "G78A and A210Q resulted in significant loss of activity supporting their essential function in catalysis and suggesting their role as an oxyanion." G78A and A210Q still have their backbone amides and thus are still possible to function as an oxyanion hole. I couldn't follow the rationale for these two mutants, especially A210Q. Oxyanion holes formed by backbone amides are usually not readily mutated (see J. Am. Chem. Soc. 2011, 133, 50, 20052-20055).
Line 207-208. "The activity of Y45A was lost about 85%, probably because Y45A mutation causes the loss of the hydrogen bond between Tyr45 and the bridge." Is this interaction between Y45 and 90-degree rotated sulfonylurea bridge present in the P44R mutant which showed increased activity? Line 388-394. The authors discussed a lot about the steric hindrance between Tyr45 and substrate while Y45A significantly reduced activity. How about Y45F mutant? Have the authors tested it? Comparison between Y45F and Y45A would tell this is true or not.
Line259. "… and no significant product inhibition was observed." The authors need to provide evidence or citation for this statement. The observation of CA complex showed better density than MM (two conformers in the structure) might suggest product inhibition.
Line 333-338 Has the authors checked whether the dihedral angles of Ile43-Pro44R fall into the allowed region of pre-Proline Ramachandran plot or not? The authors could discuss this in the manuscript.
Line 417-424. R150A showed loss of activity. The authors didn't rationalize the essential Arg150. The positively charged Arg150 could stabilize the tetrahedral acyl-enzyme intermediate.
Line 466-472. Software should be properly cited for example, XDS, PHASER, Coot, Phenix and Pymol. None has been cited. Table 1. I suppose the values in the bracket belong to the highest-resolution shell. The authors should mention it. The numbers of observed reflections and unique observations in the bracket for S209A-MM complex structure (PDB entry 7Y0L) look suspicious to me and seem contradictory to the multiplicity.
We thank the reviewers for providing constructive comments, which greatly helped us improve our manuscript. We have submitted a revised version (page numbers and lines refer to this revised version). Further we also upload a track-changes docx document showing all changes that have been made during the revision process. Please find our point-bypoint response to each of the reviewers' comments below.

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): 1. The paper is well written, although some minor English errors occur throughout and these would need to be corrected. For instance on Line 107 singular form of the verb to be (is) should be plural (are). Line 303 -has not was, Line 344 should read -Previous results.
Response: We appreciate the reviewer's positive remarks regarding our work, and thank you for pointing out some grammatical errors. We have corrected these errors. Please, see line 110, 329 and line 407. In addition, we have asked native English editors to polish and revise the manuscript.
2. There are also some inconsistencies regarding the name of the related esterase enzyme. On Line 105 the protein 1QLW is called esterase 731, while on Line 223, 226, 231 it is called esterase 713.
Response: Good catch! Esterase 731 has been corrected.
3. Table 1 would benefit from inclusion of Wilson B-factor of each data set and the clashscore of each structure.
Response: Thank you very much for your suggestion. We have followed the advice and added Wilson B-factor and the clashscore in the   3,4,3,4,. 22,23 SulE superimposed well with both esterase with a 2.0 Å root mean square deviation (RMSD) for the aligned Cα coordinates (Fig. 4a). The catalytic residues Ser, Glu and His are completely conserved (Fig. 4b).
Although the overall structure of SulE is similar to putative esterase 4Q34 and esterase 713, obvious differences were observed in the three loop regions (corresponding to lid loop, loop 110-143 and loop 240-262 in SulE) on protein surface (Fig. 4a). The lid loop of SulE is longer than those of 4Q34 and esterase 713. In addition, Loop 110-143 of SulE is also longer than that of 4Q34, whereas loop 240-262 of SulE is absent in the esterase 713….". 5. The structure reported in the submitted paper appears to be a different member of this esterase family and the reference to 1QLW should be mentioned in the introduction when the similarity is first mentioned. Response: We appreciate the reviewer for taking the time to carefully review our manuscript and providing professional feedback and constructive suggestions, which greatly help us improve the quality of this manuscript.
1. The authors wrote that a key lid loop affects substrate binding in the title, however, the manuscript lacks comprehensive study. For example, no experiments were performed to compare their binding affinity. In addition, this 21-residue lid loop (residue 31-51) contains three additional proline residues (Pro33, Pro38 and Pro41) and two glycine residues (Gly32 and Gly50). To comprehensively analyse the flexibility of the loop, the authors should analyse their impact on activity. On the other hand, the saturation mutation of Pro44 seems unnecessary and takes too much space. Mutation of Pro44 into some non-glycine residues (Ala, Asp, Cys, Glu) only showed less than 2-fold improvement and Pro44 into large polar residues (Arg, Lys, Gln) showed about 4-to 6-fold improvement. Overall, I personally think a greater improvement than an order of ten counts as significant. Are additional interactions formed in the side chains of latter mutants (Arg, Lys and Gln) in the crystal structures?
Response: We appreciate the reviewer's thoughtful suggestion.
(1). no experiments were performed to compare their binding affinity. (2). In addition, this 21-residue lid loop (residue 31-51) contains three additional proline residues (Pro33, Pro38 and Pro41) and two glycine residues (Gly32 and Gly50). To comprehensively analyse the flexibility of the loop, the authors should analyse their impact on activity.
Response: Yes, we constructed the six mutants as suggested and analyzed the effect of the mutation on the activity. The results (Fig. 7a) indicated that none of the mutations improved the enzyme activity. In fact, most of them impaired the enzyme activity, particularly the P38G mutation. This suggests that the flexibility of certain regions may be more crucial than others.

Fig. 7 Amino acid mutation analysis on lid loop. a
Effects of glycine and proline mutations in the lid loop on enzyme activity. b Effects of substitution of Pro44 by hydrophilic amino acids on enzyme activity. The relative enzyme activity of the variants to MM. Error bars represent the standard deviation from three repeats. An unpaired twotailed t-test was used to determine the statistical significance. *p < 0.01; **p < 0.001; ***p < 0.0005; ns no significance. Source data are provided as a Source Data file.
(3). Are additional interactions formed in the side chains of latter mutants (Arg, Lys and Gln) in the crystal structures?
Response: The side chain of Arginine is pointing out of the active site and exposed to protein surface, and we did not observe any interaction formed by the side chain of Arg44 with substrates ( Fig. 5c, f), suggesting that it is the loop flexibility change rather than the direct interaction of Arg44 with substrates altered the enzyme activities.   Supplementary Fig. 11c).
Superposition of S209A/H333A-TM and P44R/S209A/H333A-TM complexes revealed no significant structural differences, and also no conformational changes occurred in TM (Fig. 5h, i) WT-TM and P44R-TM (Fig. 6e, f). However, the electron density of TM is unclear, especially the sulfonylurea bridge and heterocyclic moiety (Fig. 5g) In the P44R/S209A-CA complex structure, we see a water molecule in hydrogen bonding distance with the carbonyl carbon ( Supplementary Fig. 15).
Supplementary Fig. 15 The active site of P44R/S209A-CA complex structure. The catalytic triad is shown as white sticks and CA is presented as yellow stick. A water molecule in the active site is highlighted in red. The 2Fo_Fc electron density map of CA was contoured at 1.0 σ in blue color. The distance between the water molecule and the carbonyl carbon atom is indicated by the black dotted line.

It's not clear in this manuscript whether residual activity has been seen for
S209A with MM or other substrates? They saw MM in the S209A complex structure but, they saw hydrolysed CA complex structure for CE substrate (although a CE is found in the dimer interface). MM is a better substrate for wild type SulE (2-fold) than CE (J. Agric. Food Chem. 2019, 67, 3, 836-843). My other suggestion is that, to obtain a clean fully occupied structure of CA complex, a hydrolysed product for co-crystallization is a better choice or to obtain a substrate complex, do soaking in the presence of high concentration of substrate or use a R150A or S209A/H333A for co-crystallization.
Response: We really appreciate the reviewer for this valuable suggestion. We  As R150 plays an important role in recognizing and fixing the aromatic ring of the substrate, R150 mutation may affect substrate binding. Therefore, although we obtained co-crystals of R150A with some substrates, the electron density of all seven substrates were not observed at the active site. The Validation Report of the crystal structure of R150A (PDB ID 8IW8) has also been uploaded for your review. (now is panel i), we tried to adjust the y-axis scale as much as possible to display the differences, however, due to the extremely low activity of the mutants S209A, E232A, and H33A, the image needs to be enlarged to be clearly visible. In order to better distinguish, the completely inactive mutants R150A and S209A/H333A are labeled with the letters "ND".  6. Figure 5A. Colour Tyr45 differently in apo P44R/S209A or CA bound complex structures. It's hard to tell which conformation belongs to apo or CA bound.
Response: Because we obtained the complex structure of P44R/S209A/H333A with substrates MM, CE, and TM, we have redrawn Figure 5. Comparison of the structures of Apo-P44R and P44R/S209A/H333A-substrate complexes is shown in Supplementary Fig. 11, where Tyr45 is highlighted in different colors.
The structure of P44R/S209A-CA complex is shown in Supplementary Fig. 12c. Supplementary Fig. 11 in the new manuscript is presented below for your convenience.

Supplementary Fig. 1 Sequence alignment of full-length SulE and mature SulE.
The signal peptide is marked with a cyan line, the arrow points to the signal peptide cleavage site, which is located between Ala37 and Glu38. Marked with a cyan box is the mutation position P44R or P80R.
8. Line 158-160. "As expected, mutation of Ser209, Glu232 or His333 to Ala almost completely abolished the de-esterification activity (about 0.001% activity retained), and the double mutation of Ser209 and His333 resulted in a complete loss of esterase activity ( Figure 3D), …". They authors should emphasize which substrate was used in this assay or do they all show residual activity?
Response: We appreciate this suggestion. The substrate used in this assay was MM. We modify this sentence as follows: "…As expected, mutation of Ser209, Glu232 or His333 to Ala almost completely abolished the catalytic activity towards MM (about 0.001% activity remained), and the double mutation of Ser209 and His333 resulted in a complete loss of catalytic activity towards MM (Fig. 3i),…". Response: We agree with the reviewer. Our intended meaning was to express that these two residues are important for the catalytic activity of the enzyme.
Multiple sequence alignment showed that in the esterase homologous to SulE, the amino acids corresponding to Gly78 are Ala, Ile, Leu, Phe, etc., whereas the amino acid corresponding to Ala210 are mainly Gln or His, so we selectively mutated Gly78 to Ala and Ala210 to Gln to study their effect on enzyme activity.
In order not to confuse the readers, we have deleted the A210Q mutant in the manuscript. The sentence was revised as follows: "…Accordingly, G78A mutation resulted in significant loss of activity, suggesting that maintenance of the hydrophilic environment at this position is important for SulE activity…".
10. Line 207-208. "The activity of Y45A was lost about 85%, probably because Y45A mutation causes the loss of the hydrogen bond between Tyr45 and the bridge." Is this interaction between Y45 and 90-degree rotated sulfonylurea bridge present in the P44R mutant which showed increased activity?
Response: As shown in Fig. 5c,  and Y45A would tell this is true or not.
Response: We measured the enzymatic activity of the mutant Y45F towards MM (Fig. 3i). The activity of Y45F towards MM was significantly reduced, retained only about 2% activity, which was lower than the activity of Y45A towards MM (retained 15% activity). These results indicate that Y45 has a steric hindrance effect when Pro44 is present. In contrast, increased flexibility of the loop region caused by P44R mutation eliminates the steric hindrance, because the loop region could move away when substrate binding.

Fig. 3i
The relative activity of WT SulE and its variants to MM. Error bars represent the standard deviation from three repeats. ND, not detected. Statistical analysis was performed by the two-tailed t test (*p < 0.05). Source data are provided as a Source Data file.
12. Line259. "… and no significant product inhibition was observed." The authors need to provide evidence or citation for this statement. The observation of CA complex showed better density than MM (two conformers in the structure) might suggest product inhibition.
Response: We appreciate this suggestion. As shown in Supplementary Fig. 9, there is no significant difference in Michaelis-Menten kinetics whether or not product is present. We have added citation after this sentence. Please, see line 285-286, or see below: "… and no significant product inhibition was observed ( Supplementary Fig. 9)."  Fig. 9 Inhibition effect of product CA on SulE. Michaelis-Menten kinetic experiment was performed, testing product CA at an inhibitor concentration of 1mM, while varying CE concentrations from 2 to 100 µM. Mean values for n=3 replicates ± SD are shown.
13. Line 333-338 Has the authors checked whether the dihedral angles of Ile43-Pro44R fall into the allowed region of pre-Proline Ramachandran plot or not?
The authors could discuss this in the manuscript.
Response: We appreciate this suggestion. We analyzed the pre-Proline Ramachandran plot and found that the dihedral angles of Ile43 falls into the favorite region of pre-Proline Ramachandran plot. The plot is as follows. We also discuss this in the new manuscript. Please, see line 391-397, as described below: "…Since the loop region is located at the surface of the protein, the change of Values for the outmost resolution shell are given in parentheses